EP3953681A1 - Étalonnage d'intensité de laser - Google Patents

Étalonnage d'intensité de laser

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Publication number
EP3953681A1
EP3953681A1 EP20722778.6A EP20722778A EP3953681A1 EP 3953681 A1 EP3953681 A1 EP 3953681A1 EP 20722778 A EP20722778 A EP 20722778A EP 3953681 A1 EP3953681 A1 EP 3953681A1
Authority
EP
European Patent Office
Prior art keywords
control analyte
control
characteristic
analyte
peak area
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP20722778.6A
Other languages
German (de)
English (en)
Other versions
EP3953681B1 (fr
Inventor
Erik Miller
Zhiyong Peng
James White
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Revvity Health Sciences Inc
Original Assignee
PerkinElmer Health Sciences Inc
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Filing date
Publication date
Application filed by PerkinElmer Health Sciences Inc filed Critical PerkinElmer Health Sciences Inc
Publication of EP3953681A1 publication Critical patent/EP3953681A1/fr
Application granted granted Critical
Publication of EP3953681B1 publication Critical patent/EP3953681B1/fr
Active legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
    • G01N21/274—Calibration, base line adjustment, drift correction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B01L2400/0424—Dielectrophoretic forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/12—Circuits of general importance; Signal processing
    • G01N2201/127—Calibration; base line adjustment; drift compensation
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06V—IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING
    • G06V20/00—Scenes; Scene-specific elements
    • G06V20/60—Type of objects
    • G06V20/69—Microscopic objects, e.g. biological cells or cellular parts

Definitions

  • Various aspects of the disclosure relate to calibrating the intensity of a laser performing one or more assays.
  • Electrophoresis is one technique used to measure the presence and/or concentration of a substance. Based on different migration rates for various ions, ions may be separated from each other when subjected to an electric field. Electrophoresis is used to identify concentrations of macromolecules, such as proteins, in a sample. Each run may produce slightly different results based on a number of factors including pipetting and dilution techniques, skill of the technician in preparing samples, dilution factors, numbers of runs, positioning of a well in a test system, and laser intensity, among other factors. To account for these factors, multiple runs of a sample are conducted using a set of wells with samples. The coefficient of variation (CV) is used as quality control in quantitative tests. Determining an overall CV may be complex based on combining intra-assay CVs with inter-assay CVs. Calibration of systems used to perform tests may require significant portions of time prior to the testing of samples.
  • the electrophoretic separation system may be used to separate macromolecules of sample including, but not limited to, proteins, nucleic acids, and/or other charged molecules and monitor their fluorescence when passing through a testing channel.
  • the calibration may include performing multiple runs of a known or control sample at different laser powers that can be preselected. Based on the measured data from the multiple runs using different laser powers applied to the control sample containing a control analyte, an analysis can be performed to compare the measured data from the system under test (e.g., the test system comprising the laser to be calibrated) to empirically determined data.
  • At least one characteristic of the control analyte e.g., the peak area, peak width, and/or peak height, etc.
  • a computed or calibrated laser power for the test system may be obtained by comparing the empirically determined characteristic for the control analyte, to characteristics of the control analyte that are based on measurements of the control analyte made by the test system.
  • the disclosed methods and systems include a system comprising a laser, microfluidic channel, sensor, a memory; and, a processor configured to execute instructions stored in the memory, the instructions causing the processor to: store a known concentration of a control analyte, the control analyte being present in a control sample at the known concentration; determine an empirically determined at least one characteristic of the control analyte using the known concentration of the control analyte and an empirically determined model, perform multiple tests of the control sample at different laser powers applied to the microfluidic channel, obtain measurements from the sensor while performing the multiple tests, determine the at least one characteristic of the control analyte from the sensor measurements, compute a calibrated laser power based on comparing the at least one characteristic of the control analyte from the sensor measurements and the at least one empirically determined at least one characteristic; and, perform, at the calibrated laser power, at least one test of a sample to be tested.
  • the sensor measurements are intensities of light generated by at least one of fluorescent dyes or fluorescent tags associated with components of the control sample in the microfluidic channel, and the sensor measurements may correspond to a fluorescence related to the control analyte having the known concentration.
  • the instructions may further cause the processor to store the sensor measurements.
  • the instructions to determine an empirically determined at least one characteristic of the control analyte comprise instructions to determine at least one of a peak area, peak height, or peak width based on the known concentration of the control analyte.
  • the instructions to determine an empirically determined at least one characteristic for the control analyte comprise instructions to empirically determine a peak area based on the known concentration of the control analyte and the empirical model, wherein the instructions to determine the at least one characteristic of the control analyte from the sensor measurements include instructions to compute at least one peak area based on the sensor measurements of the control analyte, and, wherein the instructions to compute a calibrated laser power include instructions to compare the empirically determined peak area with the at least one peak area determined from the sensor measurements of the control analyte, and based on the comparison, compute the calibrated laser power.
  • the instructions to determine a quadratic polynomial include instructions to determine the fitting parameters a, b, and c using at least one estimation technique, including a regression technique.
  • methods and systems that include an empirical model for a control analyte, the empirical model representing a relationship between a first characteristic of a control analyte and at least one second characteristic of the control analyte, performing multiple tests of a control sample at different laser powers, wherein the control sample comprises the control analyte with a known concentration, obtaining measurements from a sensor while performing the multiple tests, computing at least one characteristic of the control analyte based on the measurements, determining, based on the known concentration of the control analyte and the empirical model, at least one empirically determined characteristic of the control analyte, comparing the computed at least one characteristic based on the measurements with the empirically determined at least one characteristic, to provide a computed laser power; and, performing, at the computed laser
  • the empirical model is a relationship between control analyte concentration and peak area corresponding to the electrophoretic separation of the control analyte in an electrophoretic separation test, although the disclosed methods and systems are not so limited.
  • the first characteristic of the control analyte is concentration and the second characteristic of the control analyte is peak area for the control analyte in an electrophoretic separation.
  • the empirical model is a linear model, and the linear model is based on running tests of the control analyte while independently varying individual test parameters comprising at least one of concentration and laser power.
  • comparing the computed at least one characteristic of the control analyte based on the measurements with the empirically determined at least one characteristic of the control analyte includes processing the sensor measurements of the control analyte in accordance with the at least one characteristic of the control analyte that is associated with the empirical model, i.e., so that the characteristic of the control analyte derived or determined from the sensor measurements, can be utilized with the empirical model.
  • the at least one characteristic of the control analyte comprises at least one of peak area, peak height, and peak width.
  • providing an empirical model comprises collecting empirical data using a known test system while varying test parameters associated with the first characteristic and the second characteristic of the control analyte.
  • Providing an empirical model may further comprise determining a relationship between a concentration of the control analyte, and a peak area corresponding to an electrophoretic separation of the control analyte.
  • providing an empirical model comprises determining a linear relationship between a concentration of the control analyte, and a peak area corresponding to an electrophoretic separation of the control analyte.
  • Figure 1 A shows an example of a microfluidic chip tray with various channels.
  • IB shows a sample plate with various wells.
  • Figure 1C shows another sample plate with wells;
  • Figure 2 is a flow chart showing an example method for testing a sample
  • Figure 3 is a flow chart showing an example method for calibrating laser power and testing a sample
  • Figure 4 shows a graph of a line fitted through sample values and used to determine a laser power
  • Figure 5 shows an example of a computing device
  • Figure 6 shows an example of a laser illuminating a channel
  • Figures 7A and 7B show examples of laser powers for testing of samples
  • Figure 8 shows an example of a graph with results of testing a sample at multiple laser powers
  • Figure 9 shows an example of laser powers over time
  • Figure 10 shows another example of laser power over time
  • Figure 11 shows an example of multiple lasers spaced along a channel
  • Figure 12 shows examples of light intensity readings for a sample tested using four laser powers
  • Figure 13 shows an example of multiple lasers irradiating a common location along a channel
  • Figures 14A-14D show examples of laser powers spaced in time
  • Figure 15 shows a user interface permitting entry of a control sample's concentration
  • Figure 16 shows a user interface showing system status
  • Figure 17A shows an example of a reading showing area variability.
  • Figure 17B shows an example of readings taken at various laser powers.
  • Figure 17C shows another reading taken at a selected laser power;
  • Figure 18 shows variations in locations of laser irradiation per use of a microfluidic chip tray
  • Figure 19 shows an example of a laser with additional optics
  • Figure 20 shows another example of a laser with additional optics
  • Figure 21 shows a graph of examples of test runs graph of linear and non-linear regions of test runs at various laser powers
  • Figure 22 shows a graph of example areas of 120 kDa at low and high laser powers
  • Figure 23 shows a graph of different laser powers that may be used per experiment
  • Figure 24 shows an example of inter-run corrected area coefficients of variance for specific macromolecule molecular weights.
  • Figure 25 shows an example of concentration coefficients of variance for specific macromolecule molecular weights.
  • an analyte such as a macromolecule
  • concentration, and purity help ensure the efficacy and safety of macromolecule products for biopharmaceutical and other industries.
  • One technique used for measurements of macromolecules is microfluidic electrophoretic separation, the results of which can be represented by a plot of time versus intensity, with each macromolecule in a sample being associated with a “peak” on the plot, where the time of a given peak corresponds to the travel time or size/identity of a given macromolecule in the sample, and the height, size, or intensity (e.g., fluorescence measurement) of the peak can be related to a concentration of that same macromolecule.
  • proteins are used herein as an example of the analyte/macromolecules being tested, and similarly, the characteristic of the peak resulting from microfluidic electrophoretic separation that is used herein is the“peak area.” It is appreciated that other analytes or macromolecules may be tested in place of or addition to the testing of proteins, and that other characteristics of a peak may be used other than and/or in addition to peak area, e.g., peak height, peak width, etc.
  • the CV is a dimensionless number defined as the standard deviation of a set of measurements divided by the mean of the set
  • the intra-assay CV is an expression of the well-to-well consistency.
  • the inter-assay CV is an expression of the plate-to-plate consistency.
  • Running tests on multiple samples on a single plate can help account for variances in setup techniques (including pipetting techniques) of a single technician (providing a single technician prepared the samples on the plate).
  • Running tests using multiple machines using the same or different plates prepared by the same or different technicians can help account for variances in the different machines, how the same plate or different plate is placed in each machine, and different setup skills of the technicians.
  • One technique used to analyze samples is electrophoresis in which a sample is subjected to an electric field.
  • the sample is placed at one end of a channel containing a gel and an electric field applied to the gel. Based on a number of factors including, but not limited to, the electric field applied to the channel, the density of the gel, the molecular weight of a macromolecule in the sample, one or more macromolecules in the sample moves down the channel.
  • the macromolecules may be modified with one or more dyes or tags, including one or more fluorescent dyes or tags.
  • the one or more dyes or tags may be chosen to fluoresce when subjected to or interrogated by one or more selected wavelengths of light
  • the channel may be illuminated by a light source (for example, a laser or other light source) emitting the one or more wavelengths at which the one or more dyes or tags fluoresce.
  • a light source for example, a laser or other light source
  • the type and quantity of a given analyte or macromolecule may be thus determined based on (i) the time at which the dye or tag bound to the macromolecule passes (i.e., is detected at, e.g., by optical sensors) an observation area during a sample test run; and/or, (ii) the intensity of the detected fluorescence.
  • the light intensity may be related to the power applied to the light
  • one or more lasers are described as the light source.
  • other light sources may be used including fluorescent lamps, gas discharge lamps, plasma-based light sources, UV LEDs, and the like. It is appreciated that the techniques described herein may be applied to any of the various light sources to calibrate the intensity level of the light source for sample analysis purposes.
  • Figure 1 A shows an example of a microfluidic chip tray 100 with various wells, such as the PerkinElmer LabChip® product
  • the wells may include a waste well 101, wells 102 and 103 containing destain, wells 104, 105, 106, and 107 containing gel-dye, and well 108 containing a marker.
  • the wells may be connected by one or more capillaries that connect various wells.
  • a sipper capillary 109 may introduce a sample into an injection intersection 110 and be mixed with the gel-dye from one or more of wells 104, 105, and 106. Based on an applied electric field, the macromolecules in the sample travel along a separation channel 111.
  • the macromolecules may be destained from wells 102 and 103 via capillaries 112.
  • the macromolecules may be detected at location 113.
  • the number, placement, connection of the wells via capillaries, injection point or points, and detection point or points may be varied as desired.
  • An electrophoretic microchip is shown generally with dashed lines 114.
  • the microchip may include more capillaries or fewer capillaries than shown in Figure 1 A as needed.
  • Figure IB shows a sample plate 120 with various wells 121.
  • Sample plate 120 may also be referred to as a microplate, a microtiter plate, a microwell plate, or a multiwell plate
  • Wells 122 represent a column of a sample to be tested (shown crosshatched).
  • Wells 123 do not contain the sample (shown without shading or crosshatching).
  • One or more wells may contain a control sample.
  • well A12 may be the only well containing the control sample (shown shaded).
  • a full column of wells e.g., column 7 with shaded wells
  • two or more wells but less than a complete column of wells may be used to contain the control sample.
  • Other arrangements of which wells contain and do not contain a sample are possible and may vary based on the quantity of sample available, time available for testing, and the precision of the desired results.
  • the sample to be tested may be located wells other than in the column of wells 122.
  • Figure 1 C shows another sample plate 124 with wells.
  • Column 1 is a column of a sample to be tested.
  • the control sample is located in one or more wells not part of the regular set of wells.
  • the control sample may be placed in one or both of wells 125 and/or 126.
  • the size of the well or wells containing the control sample may be the same or different from other wells in the sample plate 124.
  • well 125 is larger than other wells.
  • well 126 is smaller than the other wells.
  • Other arrangements, different from those shown in Figures IB and 1C, of which wells contain and do not contain a sample are possible and may vary based on the quantity of sample available, time available for testing, and the precision of the desired results.
  • FIG. 2 is a flow chart showing an example method for testing a sample in accordance with the disclosed methods and systems.
  • step 201 one or more sample plates containing a control sample, a sample to be tested (“test sample”), and various ingredients for the tests (including for instance, gel-dye, destain solution) are loaded into a test system.
  • a known or concentration of a control analyte (e.g., macromolecule, e.g., protein) within the control sample may be identified to the system, for instance, by a technician, and from that concentration, fluorescence data can be empirically determined for that analyte (“control analyte”).
  • step 202 multiple test runs of the control sample are performed, where the laser power may be different for different runs.
  • some runs may be performed by repeating a laser power.
  • a laser power is computed based on a comparison of the measured data from the test runs on the control sample, and specifically, the control analyte, to empirically determined data for the control analyte.
  • the results from the multiple tests of the control sample are used to perform a regression analysis.
  • the regression analysis may be based on one or more of laser power (e.g., mW), observed migration time(s), observed relative fluorescence (RFU), and measured peak area(s) for the control analyte (e.g., raw data, filtered measurements, signal processed measurements, etc.).
  • the output of step 203 is a computed (calibrated) laser power that has been determined to provide a peak fluorescence and/or peak area corresponding to an empirically determined peak area, for the control analyte/macromolecule of interest.
  • step 204 using the computed laser power, tests are performed or run for the set of samples to be tested (“test samples”).
  • test parameters while collecting empirical data can vary based on the empirical model that is being determined, as the empirical data must be collected to allow for the derivation of an appropriate relationship between a first characteristic of the control analyte and a second characteristic of the control analyte.
  • a relationship e.g., model(s)
  • model(s) can be determined between the first characteristic or property of the control analyte that is associated with the control analyte, and the second control analyte characteristic or property.
  • electrophoretic separation characteristics of the control analyte can be related to control analyte characteristics or properties such as e.g., analyte concentration, laser power, etc., for the given control analyte.
  • a property or characteristic of a control analyte can be based on the control analyte or processing of data associated with the control analyte.
  • the empirical model can be represented as a linear relationship, although in other embodiments, the relationship may be represented by a quadratic or another form.
  • the relationship can be represented as a linear model (e.g., the data from the empirical test runs can be used to determine a line) between peak area (electrophoretic separation of control analyte) and associated concentration of the control analyte
  • an empirically determined peak area, y for that control analyte can be determined.
  • concentration and peak area or such other characteristic of the control analyte that can be measured by a system under test
  • FIG. 3 is a flow chart showing an example method for calibrating laser power and testing at least one sample on a given test system, once an empirical relationship/model between a control analyte and a measurement parameter has been developed and/or determined, as provided herein.
  • a known concentration of a control analyte is contained in a control sample, which may include other components such as, e.g., other proteins.
  • the control analyte can be understood to be a protein of interest, or“control protein,” and it can be understood that the control sample contains a known concentration of the control protein/analyte.
  • the control sample may also contain other analytes, e.g., other proteins and/or macromolecules.
  • the known concentration of the control protein is entered into or provided to the test systems and/or methods, and at 301 A for the example system where peak area is the characteristic of the control analyte/protein to be measured by the system under test, an empirically determined peak area may be computed based on an empirically determined model/relationship between concentration and peak area, for the control analyte/protein.
  • a model or relationship has been predetermined and/or exists prior to the entry of the known concentration by a user.
  • the concentration value of the control analyte may be entered by a technician into a user interface of the test system.
  • the concentration of the control analyte may be identified to the test system and/or method through another data entry process. For example, a technician may be prompted to scan a barcode affixed to a bottle of the control sample to obtain the concentration of the control analyte directly from the barcode or indirectly (e.g., using an internal lookup table associating barcodes with test sample concentration values).
  • a plate containing the control sample and at least one test sample is inserted into the test system.
  • the control sample and the test sample may be provided separately, e.g., in separate plates.
  • a processor selects a first laser power to be applied by a light source (e.g., laser) to the microfluidic channel.
  • a light source e.g., laser
  • the processor runs one or more tests on the control sample at the first laser power, in each case, storing the collected and corresponding data/measurements, including for example, power (e.g., of the laser or other light source), measured peak areas, and time of peaks.
  • the processor determines whether additional laser powers are to be used on the control sample.
  • the processor computes a calibrated laser power 307 based on a comparison of the measured data and the empirically determined data from 301 A.
  • a relationship between power (e.g., laser power) and peak area may be determined for the control analyte, where again, in the example embodiment, peak area corresponds to the peak area associated with the control analyte in an electrophoretic separation.
  • power e.g., laser power
  • peak area corresponds to the peak area associated with the control analyte in an electrophoretic separation.
  • measurements from the various laser powers can be analyzed specifically with respect to the control protein/analyte to provide a composite measurement (e.g., average, filtered, minimum, maximum) of peak area for the control protein at a given power.
  • Figure 4 depicts one such embodiment of a system comprising a laser, where four predetermined (4) laser powers (or power levels), e.g., approximately 2.7 mW, 3.5 mW, 4.5 mW, and 5.3 mW, were used to measure the control sample (and hence the control analyte) as provided in 303-305 of Figure 3.
  • Figure 4 depicts four (4) points, one at each of the four (4) laser powers, indicating a peak area (e.g., a composite peak area for all of the test runs at a given laser power, e.g., an average, a median, a maximum, a minimum) of the control analyte/protein as measured at the respective laser powers.
  • a peak area e.g., a composite peak area for all of the test runs at a given laser power, e.g., an average, a median, a maximum, a minimum
  • a relationship can be determined between laser power and peak area for the given test system, and the control analyte/protein.
  • the relationship can be represented by a linear, quadratic, or another relationship
  • the empirically determined peak area, 301A, for the control analyte can be used as“y” in the quadratic equation, thereby leaving a single variable “z”, or computed or calibrated laser power, to be determined. Solving or computing the quadratic equation for z, or the calibrated laser power, provides a computed or calibrated laser power for which test samples can then be run.
  • the aforementioned technique is merely of various techniques that may be used to determine the computed or calibrated laser power including, and other example methods include but are not limited to: for each set of optical/fluorescence sensor output values, find a difference between the empirically determined peak sensor for the known concentration of step 301 A and the measured peak sensor values for the control protein/analyte for one or more selected laser powers (steps 303, 306) (e.g., averaging, median, etc.), and determine the computed or calibrated laser power based on the smallest difference; or, for each set of optical/fluorescence sensor values measured, find a difference between the empirically determined peak area (301 A) for the control protein/analyte and the measured peak area of the control protein/analyte using the one or more selected laser powers (steps 303, 306), and compute the calibrated laser power based on the smallest difference; and/or, find a difference between the empirically determined peak area for the control protein/an
  • an optical/fluorescence sensor value measured above and/or an optical/fluorescence sensor value measured below and an empirically determined fluorescence of the control protein/analyte may be obtained at different selected laser powers, and, based on a ratio of the empirically determined fluorescence compared to the two fluorescence measurements, a computed calibrated laser power may be determined as between the laser powers associated with the measured fluorescence above and/or below, the empirically determined fluorescence, and relative to the measured ratio.
  • step 308 the processor sets the laser power to the computed (calibrated) laser power that was computed in step 307.
  • the processor controls the test system to use the calibrated laser power in performing subsequent tests on test samples.
  • runs of one or more sets of wells may be performed on the sample to be tested.
  • the senor for measuring fluorescence may be a CCD (charge coupled device), a CMOS sensor array, a photodiode, and/or other optical sensor.
  • CCD charge coupled device
  • CMOS complementary metal-oxide-semiconductor
  • photodiode a photodiode
  • Step 309 may be performed directly after step 308 or after a period of time and/or repeatedly at specific and/or random intervals, to (re)confirm the computation and/or selection of the calibrated laser power.
  • the processor may perform (in step 310) one or more runs using the calibrated laser power on the control sample to measure the control analyte.
  • the processor may determine whether the measured sensor values and/or data derived therefrom (e.g., time and fluorescence, or data derived therefrom, e.g., peak, area, or combination of peak and area) for the control analyte continue to approximate, to within a desired or specified tolerance, the empirically determined value(s) of the characteristic of the control analyte (e.g., peak area) determined in step 301 A. If the sensor values using the calibrated laser power remain within the specified or desired tolerance (e.g., less than 5%, less than 2 %, less than a value smaller than 1%) remain within the tolerance, then the calibrated laser power may continue 311 to be used in step 309 to perform the additional tests on samples.
  • the specified or desired tolerance e.g., less than 5%, less than 2 %, less than a value smaller than 16%
  • the processor can be instructed to re-perform the power calibration steps again starting, for example, at step 303.
  • a polynomial curve with new fitting parameters will need to be determined, and hence a new computed or calibrated laser power based on the same empirically determined characteristic of the control analyte (301 A).
  • a test system used to perform the experiments may include one or more lasers configured to illuminate a channel.
  • the channel may be part of the testing system itself or may be provided separately.
  • the channel may be part of a capillary electrophoresis microchip.
  • the microchip may have one or more integrated processors that control or assist with the control of electrophoresis testing of a sample.
  • the channel may be subjected to an electric field created between two or more electrodes charged to different potentials.
  • the computer-controlled operations described herein may be implemented in computer-readable instructions stored in memory associated with the electrophoresis microchip, in memory associated with or in a combination of both.
  • the testing system may be controlled by a computing device described in Figure 5.
  • FIG. 5 shows hardware elements of a computing device 500 that may be used to conduct the experiments described herein.
  • the computing device 500 may comprise one or more processors 501, which may execute instructions of a computer program to perform any of the functions described herein.
  • the instructions may be stored in a readonly memory (ROM) 502, random access memory (RAM) 503, removable media 504 (e.g., a USB drive, a compact disk (CD), a digital versatile disk (DVD)), and/or in any other type of computer-readable medium or memory. Instructions may also be stored in an attached (or internal) fixed drive 505 or other types of storage media.
  • ROM readonly memory
  • RAM random access memory
  • removable media 504 e.g., a USB drive, a compact disk (CD), a digital versatile disk (DVD)
  • Instructions may also be stored in an attached (or internal) fixed drive 505 or other types of storage media.
  • the computing device 500 may comprise one or more output devices, such as a display device 506 (e.g., an external display screen and/or other external or internal display device) and a speaker 511, and may comprise one or more output device controllers 507, such as a video processor.
  • One or more user input devices 508 may comprise a remote control, a keyboard, a mouse, a touch screen (which may be integrated with the display device 506), microphone, etc.
  • the computing device 500 may also comprise one or more network interfaces, such as a network input/output (I/O) interface 510 (e.g., a network card) to communicate with an external network 509.
  • I/O network input/output
  • the network I/O interface 510 may be a wired interface (e.g., electrical, RF (via coax), optical (via fiber)), a wireless interface, or a combination of the two.
  • the network I/O interface 510 may comprise a modem configured to communicate via the external network 509.
  • the computing device 500 may include a laser control circuit 512 that may cause the laser to output a beam with a specified laser power.
  • the computing device 500 may include one or more sensors 513 including, but not limited to, detecting fluorescence of a substance in the channel when irradiated by the laser.
  • the computing device 500 may include a capillary control circuit 514 that controls the operations of the capillaries of Figure 1A and provides an electrical potential to the channel.
  • the processor 501 may comprise a single processor or multiple processors where each of the multiple processors may perform fewer operations. For example, one processor may be resident in the housing containing the laser. Another processor may be resident in the microchip provided in the microfluidic chip tray of Figure 1A and control the operation of the capillaries.
  • Figure 5 shows an example hardware configuration
  • one or more of the elements of the computing device 500 may be implemented as software or a combination of hardware and software. Modifications may be made to add, remove, combine, divide, etc. components of the computing device 500. Additionally, the elements shown in Figure 5 may be implemented using basic computing devices and components that have been configured to perform operations such as are described herein.
  • a memory of the computing device 500 may store computer- executable instructions that, when executed by the processor 501 and/or one or more other processors of the computing device 500, cause the computing device 500 to perform one, some, or all of the operations described herein.
  • Such memory and processors may also or alternatively be implemented through one or more Integrated Circuits (ICs).
  • ICs Integrated Circuits
  • An IC may be, for example, a microprocessor that accesses programming instructions or other data stored in a ROM and/or hardwired into the IC.
  • an IC may comprise an Application Specific Integrated Circuit (ASIC) having gates and/or other logic dedicated to the calculations and other operations described herein.
  • ASIC Application Specific Integrated Circuit
  • An IC may perform some operations based on execution of programming instructions read from ROM or RAM, with other operations hardwired into gates or other logic. Further, an IC may be configured to output image data to a display buffer.
  • Figure 6 shows an example of a laser illuminating a channel.
  • a laser 601 generates a laser beam 605 and the beam is directed toward a channel 602.
  • both the laser and a sensor detecting a fluorescence of material in the channel 602 are shown in the same housing as part of a single optical train.
  • the sensor may be located with the laser or may be separate from the laser.
  • the laser and sensor are shown together as element 601.
  • Alignment marks 603 and 604 may optionally be provided on one or both sides of channel 602.
  • the laser irradiates spot 606. As fluorescent material passes spot 606, the material fluoresces and the fluoresce 607 is sensed by the sensor.
  • Alignment between the laser 601 and the channel 602 may affect the intensity of the fluorescence of the material in the channel.
  • the focusing of the laser on spot 606 may also affect the intensity of the fluorescence of the material.
  • FIGS 7A and 7B show examples of laser powers for testing of more than one control sample in a multiwell plate (e.g., in column 1, rows A+).
  • a well e.g., well 1A
  • four laser powers e.g., 1-4
  • the next well e.g., well IB
  • the wells are tested at a first laser power (e.g., power level 1) and then tested at a second laser power (e.g., power level 2).
  • the laser powers and the order of the wells may be varied as desired.
  • Figure 8 shows an example of a graph with results of testing a control sample at an identified concentration at multiple laser powers (e.g., with reference to Figure 3, 303, 306). Time is shown on the horizontal axis and fluorescence is shown on the vertical axis. For example, for a given control sample, known peaks appear on a range of times. As the laser power is varied (303, 306), the intensity of the fluoresce changes for the same macromolecule (i.e., time) between runs at different laser powers. In the example of Figure 8, nine different macromolecules are present in the control sample. Additionally or alternatively, a control sample of a single macromolecule may be used (for example, 120 kDa).
  • the smallest difference is the third value, corresponding to the third selected laser power.
  • the laser power associated with that third value may be selected as the calibrated laser power in step 308 of Figure 3.
  • an actual curve fitting approach may be used that would take the four measurements at 42 seconds and perform a curve fit, e.g., a linear regression, parabolic regression, etc., wherein the derived curve could be used to determine a calibrated laser power that would correspond to a peak area of 120.
  • the processor may generate a curve that fits the measured sensor values relating to the laser powers and selecting a laser power where the difference between the curve and the empirically determined sensor value is zero (or at a minimum).
  • FIG. 9 shows an example of laser powers over time.
  • four laser powers are shown and identified by laser power LP.
  • the laser powers are shown as 2.5, 4.0, 5.0, and 6.0 mW. In embodiments, these laser powers may represent relative power levels and may be modified as desired.
  • the laser powers are constant per run.
  • a laser or lasers with adjustable laser intensity outputs may be used. Additionally or alternatively, fixed light intensity outputs may be used and the intensity of the light reaching the channel may be adjusted through optics including but not limited to lenses, mirrors, prisms, adjustable apertures, and the like.
  • the identification of laser powers as ⁇ 2.5, 4.0, 5.0, and 6.0 ⁇ may be relative to an operational input or output power range of the laser or lasers.
  • a 15 mW laser may provide an adjustable power output.
  • the identified laser powers (e.g., 2, 4, 8) may be the application of a range of numbers (e.g., 0-8) applied to the range of possible power outputs capable by the laser. Additionally or alternatively, the identified laser powers may represent a specific value (e.g., 2 mW, 4, mW, 8 mW). Additionally or alternatively, the identified laser powers may be determined relative to the linear detection range of a sensor or sensors (e.g., one or more photodiodes, CCDs, CMOS sensor arrays, and the like).
  • Figure 10 shows another example of laser power versus time.
  • the laser power for a given rim is not fixed, but cycles through two or more of the laser powers.
  • the laser is cycled through four power levels in Figure 10. It is appreciated that the number of laser powers and/or levels cycled through may be modified.
  • the sensor may be synchronized with the cycling rate or run faster than the cycling rate to minimize sensing two different power levels during a given exposure of the sensor. For example, if the laser is cycling at one cycle per millisecond, the sensor may be sensing values during a window lasting one millisecond or less.
  • Figure 11 shows an example of multiple lasers spaced along a channel.
  • Lasers 1101
  • the 1102, 1103, and 1104 irradiate channel 1105 at spots 1106, 1107, 1108, and 1109, respectively.
  • the spots 1106, 1107, 1108, and 1109 are shown spaced from a reference point in the channel by distances W, X, Y, and Z, respectively.
  • the lasers 1101, 1102, 1103, and 1104 may provide the same wavelength of light but operate at different laser powers.
  • the lasers may provide light of different wavelengths and use constant or varying laser powers per run (as shown in Figures 9 and 10). Dyes that fluoresce at two or more frequencies of light may be used. Additionally or alternatively, multiple dyes that only fluoresce at one frequency may be used. Using lasers of different frequencies may reduce the total number of tests to be run. Each run may be performed with two or more lasers operating at different frequencies. Additionally or alternatively, each run may be performed with only one laser operating at a time.
  • FIG. 11 shows examples of light intensity readings for a sample tested using four laser powers where the irradiated spot from each laser was at a different location.
  • Figure 12 shows graphs of sensor readings for a control sample obtained at four laser powers (e.g., 2.5, 4.0, 5.0, and 6.0 mW). While the same control sample was used, the macromolecules arrive at different times based on the spacing of the irradiated spots.
  • the sensor values (e.g., fluorescence) for each laser may not be initially correlated to each.
  • Figure 12 shows sensor readings relating to the four lasers being time correlated with each other and combined.
  • the time value for the sensors may be adjusted by a ratio to a desired time scale (shown by a vertical, dashed line) based on the differences between the locations of the spots.
  • the desired time scale may coincide with one of the sensor readings' time scale (resulting in less than all sensor readings being adjusted) or none of the sensor readings' time scale (resulting in all sensor readings being adjusted).
  • the arrangement of Figure 11 and correlation of Figure 12 may reduce the number of times tests of a control sample are run to determine which laser power to use. If lasers 1101, 1102, 1103, and 1104 all output the same frequency of light, the laser using the selected laser power may be used for the runs of the sample to be tested. If lasers 1101, 1102, 1103, and 1104 output different frequencies of light, laser powers may be selected for each laser individually. For example, one or more of lasers 1101, 1102, 1103, or 1104 may provide light at a first wavelength (e.g., 488 ran) while another one or two of the lasers provides light at a second wavelength (e.g., 543 ran). Other light frequencies (e.g., 257 nm or 568) may be obtained by switching lasers.
  • a first wavelength e.g., 488 ran
  • another one or two of the lasers provides light at a second wavelength (e.g., 543 ran).
  • Other light frequencies e.g., 257 nm or 568) may be obtained by switching laser
  • the resulting set of sensor values from the arrangement of Figure 11 may approximate the sensor readings shown in Figure 8 (obtained using the arrangement of Figure 6) but with a reduced total testing time. For example, to obtain the sensor readings of Figure 8, at least four runs may have been performed. In contrast, to obtain the sensor readings using the arrangement of Figure 11, one run may have been performed. Additionally or alternatively, multiple rims using the arrangement of Figure 11 may provide more sensor data than that obtained per run using the arrangement of Figure 6.
  • Figure 13 shows an example of multiple lasers irradiating a common location along a channel. Macromolecules move along channel 1301 and are irradiated by lasers 1302- 1305. One or more sensors may be used to measure the fluorescence of the macromolecules at spot 1306. The one or more sensors may be combined with lasers 1302-1305 (e.g., in the same housing as or close to each laser) or may be provided separately from the lasers 1302-1305 as sensor 1307.
  • Figures 14A-14D show examples of laser powers spaced in time that may be used with the lasers of Figure 13.
  • Figures 14A-14D show graphs of a laser outputs of different lasers at different laser powers where the outputs are spaced in time.
  • the spot 1306 may receive light pulses, spaced in time, from the four lasers 1302-1305.
  • the resulting set of sensor values from the arrangement of Figure 13 may approximate the sensor readings shown in Figure 8 (obtained using the arrangement of Figure 4) but with a reduced total testing time. For example, to obtain the sensor readings of Figure 8, at least four runs may have been performed. In contrast, to obtain the sensor readings using the arrangement of Figure 13, one run (e.g., using multiple tags or dyes, with corresponding detectors and proper filters to eliminate noise/crosstalk) may have been performed. Additionally or alternatively, multiple runs using the arrangement of Figure 13 may provide more sensor data than that obtained per run using the arrangement of Figure 6.
  • Figure 15 shows a user interface permitting entry of a known or control sample's concentration.
  • Figure 15 includes a user interface 1501 including chip status information 1502 and run parameters 1503.
  • An image identifying a sample tray may be included with an identification 1504 of well locations of a control sample and an identification 1505 of well locations with a test sample.
  • well locations (not shown) may be identified that lack the control sample and lack the test sample.
  • the user interface 1501 may include a location 1506 in which a technician enters a concentration for the control sample.
  • the control sample may be located in only one well, multiple wells, or other combinations.
  • the user interface 1501 may include a region 1507 relating to a start button. Upon selection of this region 1507, the system may begin the process of conducting one or more experiments on the control sample and/or the sample to be tested.
  • Figure 16 shows a user interface showing system status.
  • the user interface 1601 may include a prime/calibration status region 1602 in which status information may be provided to a user.
  • the status information may include one or more of an identification of the test being performed, the status of the system, step time elapsed and/or remaining, run time elapsed and/or remaining, and calibration time elapsed and/or remaining.
  • the user interface 1601 may include a region 1603 in which additional calibration information may be displayed including status of the conditioning of the one or more trays, the laser(s) calibration(s), and optional verification.
  • the user interface 1601 may include a fluoresce v. time display region 1604 in which information relating to the calibration operations may be provided.
  • the information that may be provided in region 1604 may include graphs of one or more of area variability over time (Figure 17A), multiple fluoresce per laser power per time (Figure 17B) and/or fluoresce over time (Figure 17C) relating to a verification operation.
  • Figure 18 shows variations in locations of laser irradiation per use of a tray.
  • a laser/sensor 1801 is positioned relative to a channel 1802. Due to per-system alignment tolerances, technician variability, and other factors, a spot 1806 from laser beam 1805 may not be consistently aligned with the channel 1802 but may be slightly to one side or the other relative to the channel 1802 (resulting in a weaker return path 1807 fluorescence).
  • This alignment variability is shown generally as possible channel locations 1803 and 1804 (in dashed lines) relative to the laser/sensor 1801. This variability may be at least partially addressed by calibrating the laser power relative to the concentration of a known control sample as described in the disclosed methods and systems.
  • Figure 19 shows an example of a laser with additional optics that may further reduce alignment variability between an irradiated spot and a channel.
  • Figure 19 shows a laser/sensor 1901 outputting a laser beam 1905, irradiating a spot 1909. The spot 1909 is aligned relative to a channel 1902. Alternative alignments of the channel 1902 are shown as channels 1903 and 1904 (in dashed lines).
  • Figure 19 includes additional optics that may include a cylindrical or other lens or combination of lenses 1906 that provide non-spherical distortion to beam 1905 resulting in a wider (across channels 1902-1904) but not appreciably longer (along channels 1902-1904) beam 1907.
  • resulting fluorescence is condensed by lens(es) 1906 to fluorescence 1911, and sensed by the sensor associated with laser/sensor 1901.
  • Figure 20 shows another example of a laser with additional optics.
  • Figure 20 includes a laser 2001 and a channel 2002.
  • the channel 2002 may be misaligned, shown as channels 2003 and 2004 in dashed lines.
  • a non-spherical distorting lens(es) 2006 modifies laser beam 2005 into a wider but not necessarily longer beam 2007 in the direction of wider arrow 2008, irradiating spot 2009.
  • a fluorescence signal is shown generally as arrow 2010.
  • Figure 20 may include one or more half-silvered mirrors or other partially reflective optical components 2011 and/or 2015.
  • Component 2011 may reflect at least a portion of fluorescence signal 2010 (shown as arrow 2012) toward sensor 2013.
  • component 2015 may direct at least a portion (shown as arrow 2016) of fluorescence 2014 (having passed through lens(es) 2006) toward sensor 2017.
  • the use of component 2011 may reduce the effects of the distortion of lens(es) 2006 on the fluorescence signal 2010.
  • the use of component 2015 may allow sensor 2017 to be electrically and/or thermally isolated from laser 2001.
  • optical systems described herein may be modified to include one or more optical components including partially reflective components to direct or redirect a laser or fluorescence as desired. Additionally or alternatively, the optics may be moved relative to the channel to obtain a desired alignment and/or focus of the laser relative to the channel.
  • Figure 21 shows a graph of examples of test runs graph of linear and non-linear regions of test runs at various laser powers.
  • An ideal sensor is shown as the line from the origin and being linear through the full range of laser power.
  • an actual sensor may not receive enough fluorescence signal to provide an accurate sensor reading. While the actual sensor may provide a linear response for laser powers in region 2102, the actual sensor may start exhibiting a non-linear response through region 2103 and eventually become saturated at laser powers in region 2104.
  • Figure 22 shows a graph of example areas of a 120 kDa macromolecule peak at low and high laser powers.
  • the empirically determined peak area is shown as the horizontal line at 120 area.
  • the area ranged between 80-95 with an average of approximately 82.
  • the peak area ranged between 138-170 with an average of approximately 155.
  • a power lever between the high and low laser powers may be selected such that the measured area is close to the empirically determined peak area (here, 120).
  • Figure 23 shows an example of how laser powers may be modified between experiments.
  • the laser powers may be calibrated for each experiment and the laser power resulting in the closest measured peak area to the empirically determined peak area for given concentration may be used.
  • Figure 24 shows an example of inter-run corrected area coefficients of variance for specific macromolecule molecular weights of 15, 20, 29, 48, 68, and 120 kDa. Inter-run CV for corrected area approached 5% across 12 runs.
  • Figure 25 shows an example of concentration coefficients of variance for specific macromolecule molecular weights of 15, 20, 29, 48, 68, and 120 kDa. Inter-run CV for concentration approached 5% across 12 runs.

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Abstract

L'invention concerne des systèmes, des appareils et des procédés pour étalonner une puissance laser. Un système peut comprendre un échantillon à tester et un échantillon témoin qui comprend un analyte témoin. Un utilisateur peut indiquer une concentration connue de l'analyte témoin au système (par exemple, par la saisie d'une valeur de concentration dans une interface utilisateur ou un autre processus). Le système peut réaliser de multiples passes à différentes puissances laser et comparer les mesures de chaque passe avec des valeurs attendues pour l'analyte témoin à la concentration connue. À partir de cette comparaison, une puissance laser étalonnée peut être calculée et ce niveau de puissance calculé peut être utilisé par le système pour faire passer des tests sur un échantillon inconnu.
EP20722778.6A 2019-04-11 2020-04-03 Étalonnage d'intensité de laser Active EP3953681B1 (fr)

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US9377400B2 (en) * 2014-03-31 2016-06-28 Redshift Systems Corporation Motion modulation fluidic analyzer system
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